Article Antibody responses to Bordetella bronchiseptica in vaccinated and infected dogs John Ellis, Carrie Rhodes, Stacey Lacoste, Steven Krakowka Abstract — Bordetella bronchiseptica (Bb) whole cell bacterins have been replaced with acelluar vaccines. We evaluated the response to the acellular Bb vaccines in Bb-seropositive commingled laboratory beagles and clientowned dogs with various lifestyles and vaccination histories. A single parenteral dose of the acellular Bb vaccine resulted in consistent anamnestic IgG, and to a lesser, but notable extent, IgA, Bb-reactive antibody responses in the seropositive beagles. Associated with the increase in antibodies measured by enzyme-linked immunosorbent assay (ELISA) was an increase in the complement (C)-dependent IgG antibody mediated bactericidal effect on Bb in vitro. Antibody responses in client-owned dogs were more variable and were dependent upon the vaccination history and serological evidence of previous Bb exposure. Antibodies from vaccinated dogs recognized several Bb proteins, notably P68 (pertactin) and P220 (fimbrial hemagglutinin), the response to which has been shown to be disease-sparing in Bp infections. These antibody responses were similar to those in experimentally infected dogs and in dogs that had received a widely used whole cell bacterin. Résumé — Réponses des anticorps à Bordetella bronchiseptica chez des chiens vaccinés et infectés. Des bactérines de Bordetella bronchiseptica (Bb) ont été remplacées par des vaccins acellulaires. Nous avons évalué la réponse aux vaccins Bb acellulaires chez un groupe de Beagles de laboratoire séropositifs pour le Bb et des chiens de clients ayant divers styles de vie et antécédents de vaccination. Une seule dose parentérale du vaccin Bb acellulaire s’est traduite par une réponse IgG anamnestique uniforme, et à degré inférieur mais significatif, des réponses IgA et des anticorps réactifs à Bb chez les Beagles séropositifs. Une hausse des anticorps mesurés par ELISA a été accompagnée d’une augmentation de l’effet bactéricide atténué des anticorps dépendants IgG du complément (C) sur Bb in vitro. Les réponses des anticorps chez les chiens appartenant à des clients étaient plus variables et dépendaient des antécédents de vaccination et des preuves sérologiques d’une exposition antérieure à Bb. Les anticorps de chiens vaccinés reconnaissaient plusieurs protéines Bb, notamment P68 (pertactine) et P220 (hémagglutinine fimbriale), dont la réponse a été démontrée comme une protection contre la maladie lors d’une infection par Bb. Ces réponses des anticorps étaient semblables à celles des chiens infectés par expérimentation et à celles des chiens qui avaient reçu des bactérines à bacilles entiers généralement utilisés. (Traduit par Isabelle Vallières) Can Vet J 2014;55:857–864
B
Introduction
ordetella bronchiseptica (Bb), causally associated with respiratory disease in dogs and other species since the early 1900’s (1,2), is still prevalent today (3). Beginning in the late 1970’s, whole cell bacterins for parenteral delivery (4) and single component (5) and combination (6) intranasal (IN) vaccines containing modified-live Bb were developed to protect dogs
from disease associated with Bb infection. Both types of vaccines have disease-sparing efficacy in variably robust experimental challenge models of Bb-associated respiratory disease (4–7). A similar pathogen, Bordetella pertussis (Bp), the speciesspecific causative agent of whooping cough in humans, in the prevaccination era was one of the major killers in childhood (8). It is thought to have evolved from Bb, and is very closely
Department of Veterinary Microbiology (Ellis, Rhodes, Lacoste), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B4; Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, 43210, USA (Krakowka). Address all correspondence to Dr. John Ellis; e-mail: [email protected] Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. CVJ / VOL 55 / SEPTEMBER 2014
857
Table 1. Bordetella bronchiseptica-specific antibody responses before and after vaccination of client-owned dogs Dog Breed, Age (years) Last Bb Vx a
A R T I C LE
1aS 1bS 1cS 2aS 2bS 2cS 3aS 3bS 3cS 4aF 4bF 4cF 5aF 5bF
Sheltie, 5 Sheltie, 3 Labrador retriever, 6 Golden retriever, 4 Catahoula cross, 1 Pit bull cross, 3 Bernese mountain dog, 6 Great Dane, 4 Golden retriever, 6 Labrador retriever, 4 Labrador retriever, 10 Labrador retriever, 12 Terrier cross, 1 Staffordshire cross, 4
b
NA NA NA INc 6 mo ago IN 8 mo ago NA INJd 9 mo ago NA IN 9 mo ago NA NA NA INJ 9 mo ago INJ 5 mo ago
ELISAe at Vx Vaccine
ELISA at D10
D inhibitionf
13 INJ 17 0% 15 INJ 34 0% 16 None 10 38% 75 INJ 70 22% 84 INJ 117 36% 49 None 44 0% 20 INJ 87 30% 100 INJ 114 3% 80 None 79 23% 3 INJ 14 23% 0 INJ 11 0% 7 None 5 0% 46 INJ 94 0% 30 None 34 0%
a
Household number (1 to 5); dog in household (a-c); environment (S = suburban; F = rural). NA — no vaccination history available. c IN — intranasal B. bronchiseptica vaccine. d INJ — injectable B. bronchiseptica vaccine. e ELISA at Vx — units of IgG in B. bronchiseptica ELISA. f D inhibition — % increased inhibition of bacterial growth (% change in opitical density value from baseline). b
related, genetically and antigenically, to its progenitor, the primary difference being the expression of the pertussis toxin gene in Bp, but not Bb (8,9). Previewing the progressive development of parenteral vaccines for Bp in humans (8), for the purposes of refinement, reducing the potential for reactogenicity, and averting aerosol exposure of clients and owners to intranasally delivered live Bb, a prototype antigen-extract (acellular) vaccine for Bb was developed in the early 1980’s (10) and subsequently commercialized for use in dogs (10) and other target species, such as guinea pigs (11). The current acellular Bb vaccine was furthered refined in the early 1990’s. Today, acellular vaccines are the only parenteral immunogens currently used prophylactically for the relevant Bordetella spp. in both canine and human medicine in North America. In contrast to the situation with human Bp vaccines (8), and despite the frequent occurrence of Bb-associated respiratory disease and resultant common usage of vaccines for Bb in small animals, relatively little is known, or at least published, concerning the specificity and activity of antibodies induced by either natural exposure or vaccination with the commercial vaccines. The purpose of this study was to examine antibody responses, including the specificity and biological activity, stimulated in dogs by the current parenterally delivered acellular vaccine, and to compare those with responses stimulated by previously used whole cell Bb bacterin (7,12), in order to address controversy over the immunogenicity of the acellular bacterin (3,13).
Materials and methods Study populations Eight adult 2- to 3-year-old clinically normal male and female beagle dogs were group housed at the Western College of Veterinary Medicine (WCVM; Group A). The dogs had been subjects in unrelated nutrition experiments, but were not being used at the time of this study. All dogs had been vaccinated parenterally for canine “core” antigens (canine distemper virus, p arvovirus, canine adenovirus-2, and parainfluenza virus) 858
approximately annually, but had not been vaccinated recently, and had no vaccination history for Bordetella bronchiseptica. Fourteen clinically normal client-owned dogs of various ages and breeds were patients in a single veterinary practice in Calgary (Table 1; Group B). The dogs were purposely chosen, with owner consent, from multidog households, 3 suburban and 2 rural (farm), had lifestyles that allowed for potential exposure to other dogs and/or wildlife, and variable vaccination histories for Bb (IN, parenteral or no known Bb vaccines; Table 1) that would be typical of dogs in veterinary practices that are candidates for vaccination against Bb. Five dogs were from 2 farm households, 1 with 2 dogs, and 1 with 3 dogs; 9 dogs were from 3 suburban households with 3 dogs each. Banks of sera from young puppies (n = 7) and young adult kenneled beagles (n = 12) that had been vaccinated with a whole-cell Bb bacterin (Coughguard-B; Pfizer Animal Health, Kirkland, Quebec), or single component intranasal Bb vaccine (Nasasguard B, Pfizer Animal Health) in previous experiments were used for comparison (Group C). The client-owned dogs were vaccinated and bled with the consent of the owners. All dogs were maintained and handled using procedures consistent with, and approved by, the Canadian Council of Animal Care.
Study design and sampling Seven of the 8 commingled adult beagle dogs in Group A were vaccinated subcutaneously in the interscapular region on days 0 and 14 with a single component acellular Bb bacterin (Bronchicine CAe, Pfizer Animal Health); 1 of the commingled adult beagles was an unvaccinated contact control. Approximately 2 to 3 mL of blood were collected from the cephalic vein on days 0, 14, and 28. Serum was aliquoted, and stored at 220°C until used. For Group B, 2 client-owned dogs from each 3-dog household, and 1 dog from the 2-dog household, were vaccinated once as described on day 0 with the acellular bacterin; 1 dog in each household was left as an unvaccinated contact control. Serum was collected as described CVJ / VOL 55 / SEPTEMBER 2014
by the attending veterinarian in the practice on days 0 and 10 (after the single vaccination), and frozen until examined.
ELISA for B. bronchiseptica-reactive antibodies
Bacterial growth/kill assay A previously described modified C-dependent antibody- mediated bactericidal assay (14) using colorimetric measurement (15,16) was used to quantitate the growth of Bb in cultures containing canine sera. Briefly, approximately 0.5 cm of confluent growth of Bb on a blood agar plate was harvested 24 h after inoculation and was added to 10 mL of Dulbecco’s modified Eagles medium (Invitrogen Life Technologies, Burlington, Ontario) containing 100 units of guinea pig complement (Sigma-Aldrich Corp., St. Louis, Missouri, USA). Fifty microliters of the bacterial suspension were added to wells in a microtiter plate. Test sera (12.5 mL) were added in triplicate CVJ / VOL 55 / SEPTEMBER 2014
Gel electrophoresis and immunoblotting An aqueous bulk antigen preparation of the acellular vaccine (concentrated approximately 40 to 50 times compared to the commercial dose; Dr. Z. Xu, Zoetis, personal communication, 2013) was obtained from the manufacturer. Commercial acellular vaccine was obtained from the pharmacy at the WCVM and concentrated using a 10 kD centrifugation filter (Amicon Ultra-15; Millipore, Billerica, Massachusetts, USA). Protein concentrations were determined using a low concentration colorimetric dye binding assay (17) performed according to the dye manufacturer’s instructions (Bio-Rad Laboratories). The reported (lower limit) sensitivity of the assay is 8 mg/mL (Bio-Rad Laboratories). Proteins in 5 mg of ELISA antigen, 30 mL (limit of loading gel well) of vaccine bulk antigen, or 30 mL of 203 concentrated (limit of concentration) acellular vaccine were separated by sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), 12% resolving gels using a mini gel apparatus (MiniVE Gel Apparatus; GE Healthcare Life Sciences, Baie d’Urfé, Quebec) and visualized by silver staining using a commercial kit according to manufacturer’s instructions (18; Silver Stain Plus, Bio-Rad Laboratories). The reported sensitivity of the assay is 0.1 ng/mm2 (18). Separated proteins were compared to those in 5 mL of a prestained protein ladder (Benchmark Prestained Protein Ladder; Invitrogen Life Technologies), diluted 1/10 in 23 sample buffer. Stained gels were fixed for 20 min in methanol-5% acetic acid prior to being dried and photographed. For immune (Western) blots, separated proteins were electrophoretically transferred to nitrocellulose membranes (Amersham Hybond-ECL; GE Healthcare Life Sciences) using a transfer module (Amersham MiniVE Electrotransfer Unit; GE Healthcare Life Sciences). Membranes were blocked overnight at 4°C in 0.1 M PBS with 0.05% Tween 20 (PBS-T) and 3% skim milk. Representative pairs of serum samples from vaccinated and experimentally infected dogs were selected for imunoblot analysis. Dog sera were diluted 1:100 in PBS-T containing 1% skim milk (PBS-Tsm) and allowed to incubate on a strip of membrane for 90 min at room temperature (RT) on a rocking platform. The membrane strips were rinsed 3 times in TBS-Tsm and washed 3 3 10 min in TBS-Tsm. Bound antibody was detected by incubation for 90 min at RT with horseradish peroxidase (HRP)-conjugated Sheep anti-Dog IgG 859
A R T I C LE
Enzyme-linked immunosorbent assays (ELISAs) to measure IgG and IgA antibodies to B. bronchiseptica were performed as previously described (3,7,12). Briefly, 96-well flat-bottomed microtitration plates were coated overnight at 4°C with washed, sonicated B. bronchiseptica in carbonate coating buffer (7.5 mg/well). The bacterial antigen was prepared from confluent 24-h cultures of the Regina-1 isolate (7); bacteria were suspended in saline solution, and aliquots were frozen at 270°C until use. The optimal dilution of antigen had been determined in a standard checkerboard design, using serum from immune and nonimmune dogs, as positive and negative controls, respectively. The coating antigen was removed from the wells, and the plates were washed by immersion in double-distilled water containing 0.05% Tween 20. The wells were filled with phosphate-buffered saline (PBS) solution containing 0.05% Tween 20 and 1% gelatin, and plates were incubated for 30 min at 37°C. Test serum, diluted 1:50 with PBS solution containing 0.05% Tween 20 and 0.2% gelatin, was added to replicate wells. Plates were incubated at 37°C for 1 h and washed, and peroxidase-conjugated goat anti-canine IgA (Bethyl Laboratories, Montgomery, Texas, USA) or IgG (Cappel Laboratories, Cochranville, Pennsylvania, USA) diluted in PBS solution with 0.05% Tween 20, 0.2% gelatin and 4% normal goat serum was added to the wells. Plates were again incubated for 1 h and washed, and ABTS peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Maryland, USA) was added to the wells according to the manufacturer’s instructions. Reactions were stopped after 15 min at room temperature with 1% SDS (sodium dodecyl sulfate). Controls included sera from unvaccinated dogs (negative controls) and sera from dogs vaccinated against and challenged with B. bronchiseptica (positive controls); blank wells containing PBS solution with 0.05% Tween 20 and 0.2% gelatin were also used. Optical densities were read on a microplate reader (iMark; Bio-Rad Laboratories, Hercules, California, USA), using commercial software [MicroPlate Manager (MPM) 6.0; Bio-Rad Laboratories]. Optical density (OD) values were converted to ELISA units that were derived as a percentage of the OD values of test wells, compared to the OD values in wells containing positive controls. A “cut-off ” value . 15 units was previously determined to indicate seropositivity for Bb (3).
to the wells, and plates were incubated at 37°C for 24 h. The wells were then visually assessed for bacterial growth prior to the addition of 25 mL of MTT [3(4,5-dimethylthiazol-2-yl)2,5- diphenyltetrazolium bromide; Sigma-Aldrich], 5 mg/mL in distilled water. After 2 h further incubation, 50 mL of extraction buffer [12.5% sodium dodecyl sulfate, 45%NN-dimethyl formamide (grade; Sigma-Aldrich) in distilled water] were added. After 2 h incubation, the OD at 570 nm was measured using the microplate reader and the commercial software. Bacterial growth/viability (OD) in wells containing sera from before (baseline) and after vaccination were compared by calculating a percent of baseline OD to determine the percent change from baseline (change in inhibition). Controls included the assessment of Bb growth in wells containing no serum and no complement and growth in fetal calf serum with and without C.
IgA ELISA units
A R T I C LE
IgG ELISA units
Dog number
Dog number
Figure 1. Bordetella bronchiseptica (Bb)-reactive serum IgG antibody responses to vaccination with acellular Bb vaccine in Bb seropositive ($ 15 ELISA units) commingled adult laboratory beagle dogs. Seven dogs were vaccinated on days 0 and 14, and serum was collected for ELISA on days 0 (white bar), 14 (grey bar), and 28 (black bar). One commingled dog (*) was not vaccinated.
Figure 2. Bordetella bronchiseptica (Bb)-reactive serum IgA antibody responses to vaccination with acellular Bb vaccine in Bb seropositive commingled adult laboratory beagle dogs. Seven dogs were vaccinated on days 0 and 14, and serum was collected for ELISA on days 0 (white bar), 14 (grey bar), and 28 (black bar). One commingled dog (*) was not vaccinated.
(Bethyl Laboratories) diluted at 1:2000 in PBS-Tsm. After rinsing 3 times in PBS-Tsm, washing 1 3 10 min in PBS-Tsm, and washing 2 3 10 min in PBS, bound proteins were visualized using 39,39-diaminobenzidine tetrahydrochloride (DAB; Electron Microscopy Sciences, Hatfield, Pennsylvania, USA).
owned dogs in group B (Table 1). Six dogs from 2 households, 1 rural and 1 urban/suburban had no vaccination history for Bb and low baseline antibody titers on day 0 prior to vaccination. These dogs had minimal antibody responses to the vaccine (Table 1). In contrast, 2 dogs from 2 different households, with a history of vaccination with the acellular vaccine 9 mo prior to day 0 had low to moderate baseline Bb antibody titers, and 2- to 4-fold increases in Bb antibody concentrations after administration of a single dose of the acellular injectable vaccine. Three dogs with a history of IN vaccination $ 6 mo prior to day 0 had high serum antibody concentrations at the time of enlistment into the study (day 0) and did not respond considerably with increased antibodies by 10 d after administration of the injectable vaccine. None of the contact controls in the respective household had increased antibody responses to Bb during the 10-day study period.
Results Antibody responses Despite no history of vaccination against Bb, all 8 adult beagle dogs in Group A had prevaccination (baseline) IgG ELISA units $ 15, and were considered to have been previously exposed to Bb antigens (Figure 1). All of the vaccinated beagles had variably increased concentrations of Bb-reactive IgG antibodies in serum after the first dose of the acelluar vaccine, and variable, but usually less, further increased response after the second immunization (Figure 1). Similarly, 5 of the 7 vaccinated beagle dogs had variable increases in Bb-reactive IgA antibodies in serum that were less pronounced than in the IgG responses (Figure 2). One dog had a Bb-reactive IgA response that was greater than the positive control serum from a vaccinated and experimentally infected dog (Group C). This response was only minimally increased after vaccination. In contrast, the commingled unvaccinated control beagle dog had a slight decrease in IgG, and no change in IgA ELISA units over the study period. Prevaccination antibody levels and serologic responses to 1 injection of acellular vaccine were more variable in the client860
Bactericidal effects of immune sera Bactericidal activity of post-vaccination sera from dogs in group A was markedly increased (20% to 56%) compared with pre-vaccination bactericidal values and was associated with increased IgG Bb-reactive antibody concentrations in post-vaccination sera (Figures 1, 3). There was no change from baseline (day 0) in the amount of bacterial growth in wells containing serum that was collected at days 14 and 28 from the commingled unvaccinated beagle (Figure 3). In contrast, there CVJ / VOL 55 / SEPTEMBER 2014
2180 kD 2115 kD
A R T I C LE
% Baseline O.D.
282 kD
264 kD 249 kD 237 kD
226 kD
Dog number Figure 3. Antibody-dependent complement-mediated lysis of Bordetella bronchiseptica before and after vaccination (as indicated in Figure 1) of Bb-seropositive commingled beagle dogs. Bars indicate change from baseline (day 0) values using serum collected on days 14 (grey bar) and 28 (black bar). One commingled dog (*) was not vaccinated.
was more variability in the amount of bacterial killing/inhibition by the serum (antibodies) and complement using baseline and post-vaccination samples from the client-owned dogs in Group B (Table 1). In some cases changes in bacterial killing was not associated with vaccination for Bb or changes in amounts of Bb-reactive antibody as indicated by the ELISA (Table 1). In control microcultures Bb growth was reduced by approximately 50% in wells containing no serum, either Bb-seronegative canine serum, or fetal calf serum. In microcultures containing fetal calf serum, Bb, and guinea pig complement, bacterial growth was reduced by approximately 1/3 compared to similar microcultures with no complement (data not shown).
Antigen specificity of antibody responses The protein concentration of the bulk antigen was 0.032 mg/mL. No protein was detected in 20-fold concentrate of the commercial acellular vaccine using the dye staining method. Silver staining of PAGE gels revealed a wide spectrum of proteins in the Bb ELISA antigen (whole sonicated bacteria) (Figure 4). The vaccine bulk antigen preparation contained a similar constellation of proteins to that found in the ELISA antigen, while the concentrated commercial vaccine contained 5–6 faint protein bands (Figure 4). Consistently, antibodies in the serum of experimentally infected young naïve beagle puppies in group C, variably Bb-seropositive and then vaccinated laboratory beagles in group A and client-owned dogs in group B recognized or had increased (greater intensity of staining on western blots) binding to an approximately 68-kD protein (Figure 5). A second protein of approximately 220 kD was less consistently recognized by antibodies in the same serum samples (Figure 5). Several other CVJ / VOL 55 / SEPTEMBER 2014
219 kD
Figure 4. Silver stain of polyacrylamide gel electrophoresis (PAGE) gels of Bb ELISA antigen (a–5 mg protein), vaccine bulk antigen preparation (b–30 mL), and 203 concentrated commercial vaccine (c–30 mL). Lines indicate relative molecular weight of proteins in kD. Arrows indicate predicted location of pertactin (p68, thick arrow) and fimbrial hemagglutinin (p220, thin arrow).
molecules 20 to 49 kD and 75 to 100 kD were also recognized by these sera (Figure 5). These responses were similar to those found in dogs that had received the whole cell bacterin or modified-live intrasal vaccines in group C (data not shown).
Discussion Previously we reported that the acellular Bb vaccine stimulated systemic antibody responses in most dogs that received a single dose of the product upon entry to a humane society (3). The results of this study confirm and extend those observations in 2 populations of dogs in which we were better able to exclude natural exposure to Bb as being contributory to the responses. In both this and previous studies (3,12) most dogs with ELISAbased evidence of prior exposure to Bb, either naturally or by vaccination, responded with pronounced (usually greater than 2-fold) increases in antibody concentrations, consistent with typical anamnestic responses to an immunogen. In contrast, those dogs with very low or absent Bb antibody exhibited 861
A R T I C LE
D D D
D D
D
D
D
Figure 5. Immunoblots using sera from 2 representative 6-week-old puppies with low maternal antibodies before (a and c) and 14 days after (b and d) aerosol challenge with Bordetella bronchiseptica (Bb). Immunoblots using sera from representative adult Bb-low seropostitive laboratory dog before (e) and 28 days after vaccination (on days 0 and 14) with acellular Bb vaccine. Immunoblots using sera from representative adult Bb-low seropositive client-owned dog before (g) and 10 days after single vaccination (h) with acellular Bb vaccine. Lines indicate relative molecular weight of proteins in kD. Arrows indicate predicted location of pertactin (p68, thick arrow) and fimbrial hemagglutinin (p220, thin arrow).
inimal responses following administration of the acellular m Bb vaccine. This response is consistent with a primary response to a novel antigen. Both instances support the recommendation for a second or “booster” dose subsequent to a first priming dose. Together with previous work on a similar federally licensed vaccine in guinea pigs (11), our results in this study and a previous study (3) are in marked contrast with those from another group (13) that suggested that this acellular Bb vaccine is not immunogenic in dogs. Although the client-owned dogs comprised a relatively small population, there was considerable variation in their serological responses to Bb, prior to and after vaccination. The 6 seronegative dogs with no previous Bb vaccination history indicate that a lifestyle that provides potential exposure to other dogs during visits to dog parks or wildlife reservoirs of Bb infection, such 862
as naturally infected rabbits (19) does not guarantee Bb exposure and resultant seropositivity. Relatedly, such dogs that are either naive to Bb or lacking in recent exposure may respond poorly to a single dose of a bacterin, as was the case in these dogs. In contrast, dogs with moderate to high concentrations of Bb-reactive antibody by virtue of previous vaccination or exposure may not produce significantly more antibody after vaccination. This is consistent with current concepts concerning the control of antibody responses (20). In this category were 3 dogs from 2 suburban households that had high concentrations of Bb-specific antibodies 6 to 9 mo after IN vaccination. Based on the predicted kinetics of antibody rise and decay (20) and previous studies of antibody kinetics in response to a single dose of IN Bb vaccine under conditions in which antigen exposure was controlled (12), it is likely that these dogs were somehow CVJ / VOL 55 / SEPTEMBER 2014
CVJ / VOL 55 / SEPTEMBER 2014
contrast, much less is known about the antibody specificity spectrum that develops in dogs following immunogenic exposure to Bb antigens (30). Our results provide preliminary evidence that a similar consetellation of antigen specific responses occurs in dogs after exposure to Bb antigens. Notably, as in Bp-vaccinated or Bp-infected humans (8,29), pertactin, which is a major bacterial adhesion molecule and varies in size depending on the strain of Bp or Bb (8,30), appears to be immunodominant in vaccinated or infected dogs. As in the Bp model (8,29), the current acellular vaccines in the respective target species also induce pertactin-specific responses, and to a lesser extent FHA similar to those seen with the previously used whole cell bacterins. In humans, antibody responses to both proteins are associated with disease sparing (8,29). In addition, antibody responses against p68 have disease sparing effects in Bb-infected pigs (31). Given the high degree of genetic, antigenic, and biological similarity between Bp and Bb (8,9), it is highly likely that IgG responses to these proteins are associated with clinical immunity in infected dogs as well. The specific identification and role of responses to the other proteins in the acellular Bb vaccine await further investigation. In human medicine an evolution away from whole cell Bp vaccines towards acellular or subunit products was justified on the basis of adverse “vaccine reactions” (up to 50% incidence) to whole cell Bp vaccines (8). A similar trend has occurred in Bb products used for dogs. In human medicine a very successful Bp whole cell bacterin has been replaced with various subunit vaccines that contain arbitrary amounts of 3 or 4 purified monomorphic Bp proteins (8). There are population “risks” for unquestioned pursuit of “defined” immunogens at the expense of effective, but biologically more complex products such as whole cell bacterins. The recent re-emergence of whooping cough and associated disease, and even death in “at risk” human populations highlights the potential fallacy of this reductionist approach to immunization (32,33). It is now well-documented that the current acellular Bp vaccines confer a shorter duration of immunity than did the previously used whole cell bacterins (33). More ominous, is documentation of the emergence of pertactin negative virulent strains in recent outbreaks of whooping cough that have markedly decreased “susceptibility” to the immune responses induced by the current subunit Bp vaccines (34). The latter phenomenon is thought to derive, at least in part, from the altered immunogenicity and immunodominance of constituents in the current vaccines, inducing antibody responses that apparently drive bacterial evolution (34). In spite of the unspoken belief within the biologics and regulatory communities that a simple and defined immunogen is the “best” vaccine candidate, the acellular Bb vaccine product evaluated in this study is less refined than the Bp products discussed above and still contains a broad constellation of native Bordetella antigens, responses to which have been associated with disease-sparing (8,31). Our data indicate that this product induces a broader range of Bb antigen-specific responses unlike the potentially deficient responses induced in humans with comparable acellular Bp vaccines. As well, the broad range antigen specific-responses that we report here occurred in response to an acellular vaccine with very low total protein concentration and no exogenous 863
A R T I C LE
naturally exposed to Bb (or Bp) antigens in the interval between IN vaccination and enrollment in the study. Overall, the results from the client-owned dogs demonstrate the marked variation in exposure and extant response to Bordetella spp. antigens that can occur in dogs in a representative veterinary practice. Moreover, this variation also highlighted the difficulty, in the absence of a vaccination history, of predicting a given dog’s serological/ exposure status at the time of presentation for vaccination and how it is likely to respond to a “booster shot.” Several studies have reported an association between both mucosal and systemic Bb antibody responses and subsequent disease sparing in Bb infected dogs and other species (4,5,7,10, 11,21). In dogs that received either intranasal modified live Bb or a parenteral whole cell bacterin, alone or in combination, the concentration of Bb-specific mucosal IgA and serum IgG was negatively correlated with bacterial load in both the nose and oropharynx of experimentally infected dogs (7). Moreover, this disease-sparing activity of a strong IgG response to parenteral vaccination is similar to those reported for injectable vaccineinduced serological responses in humans at risk for B. pertussis infections (8,22), including the possibility that individuals that have been primed by natural (subclinical) infection respond to parenteral immunization with not just an IgG response, but also with an IgA response (23). Results obtained in the bacterial killing assay, which is an in vitro model of IgG and complement mediated “immune elimination” (24), complement those findings, although there was not complete correspondence between increases in antibody after vaccination and killing of bacteria in vitro. It is likely that antibodies that bind to the 68-kD protein (pertactin), are responsible for bactericidal activity (25,26). The variability that we observed in antibody/C-mediated killing likely reflects the reported complexity of Bp-complement interactions that are described in humans (26). Both Bp and Bb possess a virulence factor, BrkA, a 103 kD transmembrane protein. This protein inhibits complement activation after binding/inactivating activated complement component C1, critical to completion of complement activation via the classical complement pathway (27). Curiously, some individuals mount an immune response to this protein that counteracts this effect, others do not (27,28). High concentrations of Bordetella-reactive IgA or subtypes of IgG, which do not activate complement can also “compete” for binding sites on bacterial surfaces, thereby blocking the IgG-C killing (28). These IgG subclass or IgA blocking effects are dependent upon the concentrations of the particular antibodies, and may vary with individual immune responses to vaccination versus infection (28). Whether these mechanisms play a role in modulating Bb bactericidal activity in dogs requires further investigation. In humans, the antigen specificity of the Bp-reactive antibody responses to infection and vaccination is well-documented (8,22,29). These responses depend somewhat on the formulation of the vaccine used in the case of vaccinates. A typical human response to Bp includes the development of antibodies that bind to the pertussis toxin (not expressed in Bb), the 220-kD fimbrial hemagglutinin (FHA), the 68 to 69-kD pertactin (PRN), various fimbrial antigens, the adenylate cyclase toxin, Bp lipopolysaccharide, unidentified molecules of 75 and 84 kD (8,29). In
adjuvant. These unique features should minimize the potential for adverse reactions to this vaccine when administered alone or in combination with other vaccines (35,36).
A R T I C LE
Acknowledgments The authors thank Dr. Yolande Miles and colleagues at the Due South Animal Hospital, Calgary, Alberta, for the provision of sera from client-owned dogs. This work was supported by the principal author’s discretionary funds to study viral immunology. CVJ
References 1. Ferry NS. A preliminary report of bacterial findings in canine distemper. Am Vet Rev 1910;37:499–504. 2. M’Gowan JP. Some observations on a laboratory epidemic, principally among dogs and cats in which the animals affected presented the symptoms of the disease called “distemper.” J Pathol Bacteriol 1911; 15:372–426. 3. Ellis JA, Anseeuw E, Gow S, et al. Seroepidemiology of respiratory (group 2) canine coronavirus, canine parainfluenza virus and Bordetella bronchiseptica infections in urban dogs in a humane shelter and in rural dogs in small communities. Can Vet J 2011;52:861–868. 4. McCandlish IA, Thompson H. Vaccination against canine bordetellosis using an aluminum hydroxide adjuvant vaccine. Res Vet Sci 1978; 25:51–57. 5. Shade FJ, Goodnow RA. Intranasal immunization of dogs against Bordetella bronchiseptica-induced tracheobronchitis (kennel cough) with modified-live Bordetella bronchiseptica vaccine. Am J Vet Res 1979;40:1241–1243. 6. Kontor EJ, Wegryzn MA, Goodnow RA. Canine infectious trachobronchitis: Effects of an intranasal live canine parainfluenza-Bordetella bronchiseptica vaccine on viral shedding and clinical tracheobronchitis (kennel cough). Am J Vet Res 1981;42:1694–1698. 7. Ellis JA, Haines DM, West KH, et al. Effect of vaccination on experimental infection with Bordetella bronchiseptica dogs. J Am Vet Med Assoc 2001;218:367–375. 8. Mattoo S, Cherry JD. Molecular pathogenesis, epidemiology, clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Micro Rev 2005;18:326–382. 9. Bjornstad ON, Harvill ET. Evolution and emergence of Bordetella in humans. Trends in Microbiol 2005;13:355–359. 10. Shade FJ, Rapp VJ. Studies of a bacterin incorporating an extracted Bordetella bronchiseptica antigen for controlling canine bordetellosis. Vet Med/Sm Anim Clin 1982:1635–1639. 11. Stephenson EH, Trahan CJ, Ezzell JW, et al. Efficacy of a commercial bacterin in protecting Strain 13 guineapigs against Bordetella bronchiseptica pneumonia. Lab An 1989;23:261–269. 12. Ellis JA, Krakowka GS, Dayton AD, Konoby C. Comparative efficacy of an injectable vaccine and an intranasal vaccine in stimulating Bordetella bronchiseptica-reactive antibody responses in seropositive dogs. J Am Vet Med Assoc 2002;220:43–48. 13. Davis R, Jayappa H, Abdelmagid OY, Armstrong R, Sweeney D, Lehr C. Comparison of the mucosal immune response in dogs vaccinated with either an intranasal avirulent live culture or a subcutaneous antigen extract vaccine of Bordetella bronchiseptica. Vet Therapeutics 2007;8:32–40. 14. Callister SM, Jobe DA, Agger WA, et al. Ability of the borreliacidal antibody test to confirm Lyme disease in clinical practice. Clin Diagn Lab Immunol 2002;9:908–912. 15. Stevens MG, Kehrli ME, Canning PC. A colorimetric assay for quantitating bovine neutrophil bactericidal activity. Vet Immunol Immunpathol 1991;28:45–56.
864
16. Ellis JA, Hawk DA, Mills KW, Pratt DL. Antigen specificity and activity of ovine antibodies induced by immunization with Corynebacterium pseudotuberculosis culture filtrate. Vet Immunol Immunopathol 1991;28:303–316. 17. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254. 18. Switzer RC, Merril CR, Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem 1979;98:231–237. 19. Pathak AK, Creppage KE, Werner JR, Cattadori IM. Immune regulation of a chronic bacterial infection and consequences for pathogen transmission. BMC Microbiol 2010;10:226. 20. Tizzard IR. B cells and their response to antigens. In: Veterinary Immunology: An Introduction. 8th ed. St. Louis, Missouri: Saunders Elsevier, 2009:152–169. 21. Goodnow RA, Shade FJ, Switzer WP. Efficacy of Bordetella bronchiseptica bacterin in controlling enzootic atrophic rhinitis in swine. Am J Vet Res 1979;40:58–60. 22. Cherry JD, Gorbein J, Heininger U, Stehr K. A search for serological correlates of immunity to Bordetella pertussis cough illness. Vaccine 1998;16:1901–1906. 23. Le T, Cherry JD, Chang SJ, et al. Immune responses and antibody decay following immunization of adolescents and adults with an acellular pertussis vaccine: APERT study. J Infect Dis 2004;190:535–544. 24. Tizzard IR. Immunity at body surfaces. In: Veterinary Immunology: An Introduction. 8th ed. St. Louis, Missouri: Saunders Elsevier, 2009:245–248. 25. Gotto JW, Eckhardt T, Reilly PA. Biochemical and immunological properties of two forms of pertactin, the 69 000-molecularweight outer membrane protein of Bordetella pertussis. Infect Immun 1993;61:2211–2215. 26. Weiss AA, Patton AK, Millen SH, Chang SJ, Ward JI, Bernstein DI. Acellular pertussis vaccines and complement killing of Bordetella pertussis. Infect Immun 2004;72:7346–7351. 27. Barnes MG, Weiss AA. BrkA protein of Bordetella pertussis inhibits the classical pathway of complement after C1 deposition. Infect Immun 2001;69:3067–3072. 28. Griffiss JM, Goroff DK. IgA blocks IgM and IgG-initiated immune lysis by separate moleucular mechanisms. J Immunol 1983;130:2882–2885. 29. Redd SC, Rumschlag HS, Biellik RJ, Sanden GN, Reimer CB, Cohen ML. Immunoblot analysis of humoral immune responses following infection with Bordetella pertussis or immunization with diphtheriatetanus-pertussis vaccine. J Clin Microbiol 1988;26:1373–1377. 30. Keil DJ, Fenwick B. Strain- and growth condition-dependent variability in outer membrane protein expression by Bordetella bronchiseptica isolates from dogs. Am J Vet Res 1999;60:1016–1021. 31. Kobisch M, Novotny P. Identification of a 68-kilodalton outer membrane protein as the major protective antigen of Bordetella bronchiseptica by using specific-pathogen-free piglets. Infect Immun 1990;58:352–357. 32. Cherry JD. Epidemic pertussis in 2012 — The resurgence of a vaccinepreventable disease. New Eng J Med 2012;367:785–787. 33. Klein NP, Bartlett J, Rowhani-Rahbar A, Fireman B, Baxter R. Waning protection after fifth dose of acellular pertussis vaccine in children. New Eng J Med 2012;367:1013–1019. 34. Queenan AM, Cassiday PK, Evangelista A. Pertactin-negative variants of Bordetella pertussis in the United States. New Eng J Med 2012;368:583–584. 35. Moore GE, Guptil LF, Ward MP, et al. Adverse events diagnosed within three days of vaccine administration in dogs. J Am Vet Med Assoc 2007; 227:1102–1108. 36. Tizzard IR. The use of vaccines. In: Veterinary Immunology: An Introduction. 8th ed. St. Louis, Missouri: Saunders Elsevier, 2009: 270–276.
CVJ / VOL 55 / SEPTEMBER 2014
B
Introduction
ordetella bronchiseptica (Bb), causally associated with respiratory disease in dogs and other species since the early 1900’s (1,2), is still prevalent today (3). Beginning in the late 1970’s, whole cell bacterins for parenteral delivery (4) and single component (5) and combination (6) intranasal (IN) vaccines containing modified-live Bb were developed to protect dogs
from disease associated with Bb infection. Both types of vaccines have disease-sparing efficacy in variably robust experimental challenge models of Bb-associated respiratory disease (4–7). A similar pathogen, Bordetella pertussis (Bp), the speciesspecific causative agent of whooping cough in humans, in the prevaccination era was one of the major killers in childhood (8). It is thought to have evolved from Bb, and is very closely
Department of Veterinary Microbiology (Ellis, Rhodes, Lacoste), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B4; Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, 43210, USA (Krakowka). Address all correspondence to Dr. John Ellis; e-mail: [email protected] Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. CVJ / VOL 55 / SEPTEMBER 2014
857
Table 1. Bordetella bronchiseptica-specific antibody responses before and after vaccination of client-owned dogs Dog Breed, Age (years) Last Bb Vx a
A R T I C LE
1aS 1bS 1cS 2aS 2bS 2cS 3aS 3bS 3cS 4aF 4bF 4cF 5aF 5bF
Sheltie, 5 Sheltie, 3 Labrador retriever, 6 Golden retriever, 4 Catahoula cross, 1 Pit bull cross, 3 Bernese mountain dog, 6 Great Dane, 4 Golden retriever, 6 Labrador retriever, 4 Labrador retriever, 10 Labrador retriever, 12 Terrier cross, 1 Staffordshire cross, 4
b
NA NA NA INc 6 mo ago IN 8 mo ago NA INJd 9 mo ago NA IN 9 mo ago NA NA NA INJ 9 mo ago INJ 5 mo ago
ELISAe at Vx Vaccine
ELISA at D10
D inhibitionf
13 INJ 17 0% 15 INJ 34 0% 16 None 10 38% 75 INJ 70 22% 84 INJ 117 36% 49 None 44 0% 20 INJ 87 30% 100 INJ 114 3% 80 None 79 23% 3 INJ 14 23% 0 INJ 11 0% 7 None 5 0% 46 INJ 94 0% 30 None 34 0%
a
Household number (1 to 5); dog in household (a-c); environment (S = suburban; F = rural). NA — no vaccination history available. c IN — intranasal B. bronchiseptica vaccine. d INJ — injectable B. bronchiseptica vaccine. e ELISA at Vx — units of IgG in B. bronchiseptica ELISA. f D inhibition — % increased inhibition of bacterial growth (% change in opitical density value from baseline). b
related, genetically and antigenically, to its progenitor, the primary difference being the expression of the pertussis toxin gene in Bp, but not Bb (8,9). Previewing the progressive development of parenteral vaccines for Bp in humans (8), for the purposes of refinement, reducing the potential for reactogenicity, and averting aerosol exposure of clients and owners to intranasally delivered live Bb, a prototype antigen-extract (acellular) vaccine for Bb was developed in the early 1980’s (10) and subsequently commercialized for use in dogs (10) and other target species, such as guinea pigs (11). The current acellular Bb vaccine was furthered refined in the early 1990’s. Today, acellular vaccines are the only parenteral immunogens currently used prophylactically for the relevant Bordetella spp. in both canine and human medicine in North America. In contrast to the situation with human Bp vaccines (8), and despite the frequent occurrence of Bb-associated respiratory disease and resultant common usage of vaccines for Bb in small animals, relatively little is known, or at least published, concerning the specificity and activity of antibodies induced by either natural exposure or vaccination with the commercial vaccines. The purpose of this study was to examine antibody responses, including the specificity and biological activity, stimulated in dogs by the current parenterally delivered acellular vaccine, and to compare those with responses stimulated by previously used whole cell Bb bacterin (7,12), in order to address controversy over the immunogenicity of the acellular bacterin (3,13).
Materials and methods Study populations Eight adult 2- to 3-year-old clinically normal male and female beagle dogs were group housed at the Western College of Veterinary Medicine (WCVM; Group A). The dogs had been subjects in unrelated nutrition experiments, but were not being used at the time of this study. All dogs had been vaccinated parenterally for canine “core” antigens (canine distemper virus, p arvovirus, canine adenovirus-2, and parainfluenza virus) 858
approximately annually, but had not been vaccinated recently, and had no vaccination history for Bordetella bronchiseptica. Fourteen clinically normal client-owned dogs of various ages and breeds were patients in a single veterinary practice in Calgary (Table 1; Group B). The dogs were purposely chosen, with owner consent, from multidog households, 3 suburban and 2 rural (farm), had lifestyles that allowed for potential exposure to other dogs and/or wildlife, and variable vaccination histories for Bb (IN, parenteral or no known Bb vaccines; Table 1) that would be typical of dogs in veterinary practices that are candidates for vaccination against Bb. Five dogs were from 2 farm households, 1 with 2 dogs, and 1 with 3 dogs; 9 dogs were from 3 suburban households with 3 dogs each. Banks of sera from young puppies (n = 7) and young adult kenneled beagles (n = 12) that had been vaccinated with a whole-cell Bb bacterin (Coughguard-B; Pfizer Animal Health, Kirkland, Quebec), or single component intranasal Bb vaccine (Nasasguard B, Pfizer Animal Health) in previous experiments were used for comparison (Group C). The client-owned dogs were vaccinated and bled with the consent of the owners. All dogs were maintained and handled using procedures consistent with, and approved by, the Canadian Council of Animal Care.
Study design and sampling Seven of the 8 commingled adult beagle dogs in Group A were vaccinated subcutaneously in the interscapular region on days 0 and 14 with a single component acellular Bb bacterin (Bronchicine CAe, Pfizer Animal Health); 1 of the commingled adult beagles was an unvaccinated contact control. Approximately 2 to 3 mL of blood were collected from the cephalic vein on days 0, 14, and 28. Serum was aliquoted, and stored at 220°C until used. For Group B, 2 client-owned dogs from each 3-dog household, and 1 dog from the 2-dog household, were vaccinated once as described on day 0 with the acellular bacterin; 1 dog in each household was left as an unvaccinated contact control. Serum was collected as described CVJ / VOL 55 / SEPTEMBER 2014
by the attending veterinarian in the practice on days 0 and 10 (after the single vaccination), and frozen until examined.
ELISA for B. bronchiseptica-reactive antibodies
Bacterial growth/kill assay A previously described modified C-dependent antibody- mediated bactericidal assay (14) using colorimetric measurement (15,16) was used to quantitate the growth of Bb in cultures containing canine sera. Briefly, approximately 0.5 cm of confluent growth of Bb on a blood agar plate was harvested 24 h after inoculation and was added to 10 mL of Dulbecco’s modified Eagles medium (Invitrogen Life Technologies, Burlington, Ontario) containing 100 units of guinea pig complement (Sigma-Aldrich Corp., St. Louis, Missouri, USA). Fifty microliters of the bacterial suspension were added to wells in a microtiter plate. Test sera (12.5 mL) were added in triplicate CVJ / VOL 55 / SEPTEMBER 2014
Gel electrophoresis and immunoblotting An aqueous bulk antigen preparation of the acellular vaccine (concentrated approximately 40 to 50 times compared to the commercial dose; Dr. Z. Xu, Zoetis, personal communication, 2013) was obtained from the manufacturer. Commercial acellular vaccine was obtained from the pharmacy at the WCVM and concentrated using a 10 kD centrifugation filter (Amicon Ultra-15; Millipore, Billerica, Massachusetts, USA). Protein concentrations were determined using a low concentration colorimetric dye binding assay (17) performed according to the dye manufacturer’s instructions (Bio-Rad Laboratories). The reported (lower limit) sensitivity of the assay is 8 mg/mL (Bio-Rad Laboratories). Proteins in 5 mg of ELISA antigen, 30 mL (limit of loading gel well) of vaccine bulk antigen, or 30 mL of 203 concentrated (limit of concentration) acellular vaccine were separated by sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), 12% resolving gels using a mini gel apparatus (MiniVE Gel Apparatus; GE Healthcare Life Sciences, Baie d’Urfé, Quebec) and visualized by silver staining using a commercial kit according to manufacturer’s instructions (18; Silver Stain Plus, Bio-Rad Laboratories). The reported sensitivity of the assay is 0.1 ng/mm2 (18). Separated proteins were compared to those in 5 mL of a prestained protein ladder (Benchmark Prestained Protein Ladder; Invitrogen Life Technologies), diluted 1/10 in 23 sample buffer. Stained gels were fixed for 20 min in methanol-5% acetic acid prior to being dried and photographed. For immune (Western) blots, separated proteins were electrophoretically transferred to nitrocellulose membranes (Amersham Hybond-ECL; GE Healthcare Life Sciences) using a transfer module (Amersham MiniVE Electrotransfer Unit; GE Healthcare Life Sciences). Membranes were blocked overnight at 4°C in 0.1 M PBS with 0.05% Tween 20 (PBS-T) and 3% skim milk. Representative pairs of serum samples from vaccinated and experimentally infected dogs were selected for imunoblot analysis. Dog sera were diluted 1:100 in PBS-T containing 1% skim milk (PBS-Tsm) and allowed to incubate on a strip of membrane for 90 min at room temperature (RT) on a rocking platform. The membrane strips were rinsed 3 times in TBS-Tsm and washed 3 3 10 min in TBS-Tsm. Bound antibody was detected by incubation for 90 min at RT with horseradish peroxidase (HRP)-conjugated Sheep anti-Dog IgG 859
A R T I C LE
Enzyme-linked immunosorbent assays (ELISAs) to measure IgG and IgA antibodies to B. bronchiseptica were performed as previously described (3,7,12). Briefly, 96-well flat-bottomed microtitration plates were coated overnight at 4°C with washed, sonicated B. bronchiseptica in carbonate coating buffer (7.5 mg/well). The bacterial antigen was prepared from confluent 24-h cultures of the Regina-1 isolate (7); bacteria were suspended in saline solution, and aliquots were frozen at 270°C until use. The optimal dilution of antigen had been determined in a standard checkerboard design, using serum from immune and nonimmune dogs, as positive and negative controls, respectively. The coating antigen was removed from the wells, and the plates were washed by immersion in double-distilled water containing 0.05% Tween 20. The wells were filled with phosphate-buffered saline (PBS) solution containing 0.05% Tween 20 and 1% gelatin, and plates were incubated for 30 min at 37°C. Test serum, diluted 1:50 with PBS solution containing 0.05% Tween 20 and 0.2% gelatin, was added to replicate wells. Plates were incubated at 37°C for 1 h and washed, and peroxidase-conjugated goat anti-canine IgA (Bethyl Laboratories, Montgomery, Texas, USA) or IgG (Cappel Laboratories, Cochranville, Pennsylvania, USA) diluted in PBS solution with 0.05% Tween 20, 0.2% gelatin and 4% normal goat serum was added to the wells. Plates were again incubated for 1 h and washed, and ABTS peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Maryland, USA) was added to the wells according to the manufacturer’s instructions. Reactions were stopped after 15 min at room temperature with 1% SDS (sodium dodecyl sulfate). Controls included sera from unvaccinated dogs (negative controls) and sera from dogs vaccinated against and challenged with B. bronchiseptica (positive controls); blank wells containing PBS solution with 0.05% Tween 20 and 0.2% gelatin were also used. Optical densities were read on a microplate reader (iMark; Bio-Rad Laboratories, Hercules, California, USA), using commercial software [MicroPlate Manager (MPM) 6.0; Bio-Rad Laboratories]. Optical density (OD) values were converted to ELISA units that were derived as a percentage of the OD values of test wells, compared to the OD values in wells containing positive controls. A “cut-off ” value . 15 units was previously determined to indicate seropositivity for Bb (3).
to the wells, and plates were incubated at 37°C for 24 h. The wells were then visually assessed for bacterial growth prior to the addition of 25 mL of MTT [3(4,5-dimethylthiazol-2-yl)2,5- diphenyltetrazolium bromide; Sigma-Aldrich], 5 mg/mL in distilled water. After 2 h further incubation, 50 mL of extraction buffer [12.5% sodium dodecyl sulfate, 45%NN-dimethyl formamide (grade; Sigma-Aldrich) in distilled water] were added. After 2 h incubation, the OD at 570 nm was measured using the microplate reader and the commercial software. Bacterial growth/viability (OD) in wells containing sera from before (baseline) and after vaccination were compared by calculating a percent of baseline OD to determine the percent change from baseline (change in inhibition). Controls included the assessment of Bb growth in wells containing no serum and no complement and growth in fetal calf serum with and without C.
IgA ELISA units
A R T I C LE
IgG ELISA units
Dog number
Dog number
Figure 1. Bordetella bronchiseptica (Bb)-reactive serum IgG antibody responses to vaccination with acellular Bb vaccine in Bb seropositive ($ 15 ELISA units) commingled adult laboratory beagle dogs. Seven dogs were vaccinated on days 0 and 14, and serum was collected for ELISA on days 0 (white bar), 14 (grey bar), and 28 (black bar). One commingled dog (*) was not vaccinated.
Figure 2. Bordetella bronchiseptica (Bb)-reactive serum IgA antibody responses to vaccination with acellular Bb vaccine in Bb seropositive commingled adult laboratory beagle dogs. Seven dogs were vaccinated on days 0 and 14, and serum was collected for ELISA on days 0 (white bar), 14 (grey bar), and 28 (black bar). One commingled dog (*) was not vaccinated.
(Bethyl Laboratories) diluted at 1:2000 in PBS-Tsm. After rinsing 3 times in PBS-Tsm, washing 1 3 10 min in PBS-Tsm, and washing 2 3 10 min in PBS, bound proteins were visualized using 39,39-diaminobenzidine tetrahydrochloride (DAB; Electron Microscopy Sciences, Hatfield, Pennsylvania, USA).
owned dogs in group B (Table 1). Six dogs from 2 households, 1 rural and 1 urban/suburban had no vaccination history for Bb and low baseline antibody titers on day 0 prior to vaccination. These dogs had minimal antibody responses to the vaccine (Table 1). In contrast, 2 dogs from 2 different households, with a history of vaccination with the acellular vaccine 9 mo prior to day 0 had low to moderate baseline Bb antibody titers, and 2- to 4-fold increases in Bb antibody concentrations after administration of a single dose of the acellular injectable vaccine. Three dogs with a history of IN vaccination $ 6 mo prior to day 0 had high serum antibody concentrations at the time of enlistment into the study (day 0) and did not respond considerably with increased antibodies by 10 d after administration of the injectable vaccine. None of the contact controls in the respective household had increased antibody responses to Bb during the 10-day study period.
Results Antibody responses Despite no history of vaccination against Bb, all 8 adult beagle dogs in Group A had prevaccination (baseline) IgG ELISA units $ 15, and were considered to have been previously exposed to Bb antigens (Figure 1). All of the vaccinated beagles had variably increased concentrations of Bb-reactive IgG antibodies in serum after the first dose of the acelluar vaccine, and variable, but usually less, further increased response after the second immunization (Figure 1). Similarly, 5 of the 7 vaccinated beagle dogs had variable increases in Bb-reactive IgA antibodies in serum that were less pronounced than in the IgG responses (Figure 2). One dog had a Bb-reactive IgA response that was greater than the positive control serum from a vaccinated and experimentally infected dog (Group C). This response was only minimally increased after vaccination. In contrast, the commingled unvaccinated control beagle dog had a slight decrease in IgG, and no change in IgA ELISA units over the study period. Prevaccination antibody levels and serologic responses to 1 injection of acellular vaccine were more variable in the client860
Bactericidal effects of immune sera Bactericidal activity of post-vaccination sera from dogs in group A was markedly increased (20% to 56%) compared with pre-vaccination bactericidal values and was associated with increased IgG Bb-reactive antibody concentrations in post-vaccination sera (Figures 1, 3). There was no change from baseline (day 0) in the amount of bacterial growth in wells containing serum that was collected at days 14 and 28 from the commingled unvaccinated beagle (Figure 3). In contrast, there CVJ / VOL 55 / SEPTEMBER 2014
2180 kD 2115 kD
A R T I C LE
% Baseline O.D.
282 kD
264 kD 249 kD 237 kD
226 kD
Dog number Figure 3. Antibody-dependent complement-mediated lysis of Bordetella bronchiseptica before and after vaccination (as indicated in Figure 1) of Bb-seropositive commingled beagle dogs. Bars indicate change from baseline (day 0) values using serum collected on days 14 (grey bar) and 28 (black bar). One commingled dog (*) was not vaccinated.
was more variability in the amount of bacterial killing/inhibition by the serum (antibodies) and complement using baseline and post-vaccination samples from the client-owned dogs in Group B (Table 1). In some cases changes in bacterial killing was not associated with vaccination for Bb or changes in amounts of Bb-reactive antibody as indicated by the ELISA (Table 1). In control microcultures Bb growth was reduced by approximately 50% in wells containing no serum, either Bb-seronegative canine serum, or fetal calf serum. In microcultures containing fetal calf serum, Bb, and guinea pig complement, bacterial growth was reduced by approximately 1/3 compared to similar microcultures with no complement (data not shown).
Antigen specificity of antibody responses The protein concentration of the bulk antigen was 0.032 mg/mL. No protein was detected in 20-fold concentrate of the commercial acellular vaccine using the dye staining method. Silver staining of PAGE gels revealed a wide spectrum of proteins in the Bb ELISA antigen (whole sonicated bacteria) (Figure 4). The vaccine bulk antigen preparation contained a similar constellation of proteins to that found in the ELISA antigen, while the concentrated commercial vaccine contained 5–6 faint protein bands (Figure 4). Consistently, antibodies in the serum of experimentally infected young naïve beagle puppies in group C, variably Bb-seropositive and then vaccinated laboratory beagles in group A and client-owned dogs in group B recognized or had increased (greater intensity of staining on western blots) binding to an approximately 68-kD protein (Figure 5). A second protein of approximately 220 kD was less consistently recognized by antibodies in the same serum samples (Figure 5). Several other CVJ / VOL 55 / SEPTEMBER 2014
219 kD
Figure 4. Silver stain of polyacrylamide gel electrophoresis (PAGE) gels of Bb ELISA antigen (a–5 mg protein), vaccine bulk antigen preparation (b–30 mL), and 203 concentrated commercial vaccine (c–30 mL). Lines indicate relative molecular weight of proteins in kD. Arrows indicate predicted location of pertactin (p68, thick arrow) and fimbrial hemagglutinin (p220, thin arrow).
molecules 20 to 49 kD and 75 to 100 kD were also recognized by these sera (Figure 5). These responses were similar to those found in dogs that had received the whole cell bacterin or modified-live intrasal vaccines in group C (data not shown).
Discussion Previously we reported that the acellular Bb vaccine stimulated systemic antibody responses in most dogs that received a single dose of the product upon entry to a humane society (3). The results of this study confirm and extend those observations in 2 populations of dogs in which we were better able to exclude natural exposure to Bb as being contributory to the responses. In both this and previous studies (3,12) most dogs with ELISAbased evidence of prior exposure to Bb, either naturally or by vaccination, responded with pronounced (usually greater than 2-fold) increases in antibody concentrations, consistent with typical anamnestic responses to an immunogen. In contrast, those dogs with very low or absent Bb antibody exhibited 861
A R T I C LE
D D D
D D
D
D
D
Figure 5. Immunoblots using sera from 2 representative 6-week-old puppies with low maternal antibodies before (a and c) and 14 days after (b and d) aerosol challenge with Bordetella bronchiseptica (Bb). Immunoblots using sera from representative adult Bb-low seropostitive laboratory dog before (e) and 28 days after vaccination (on days 0 and 14) with acellular Bb vaccine. Immunoblots using sera from representative adult Bb-low seropositive client-owned dog before (g) and 10 days after single vaccination (h) with acellular Bb vaccine. Lines indicate relative molecular weight of proteins in kD. Arrows indicate predicted location of pertactin (p68, thick arrow) and fimbrial hemagglutinin (p220, thin arrow).
inimal responses following administration of the acellular m Bb vaccine. This response is consistent with a primary response to a novel antigen. Both instances support the recommendation for a second or “booster” dose subsequent to a first priming dose. Together with previous work on a similar federally licensed vaccine in guinea pigs (11), our results in this study and a previous study (3) are in marked contrast with those from another group (13) that suggested that this acellular Bb vaccine is not immunogenic in dogs. Although the client-owned dogs comprised a relatively small population, there was considerable variation in their serological responses to Bb, prior to and after vaccination. The 6 seronegative dogs with no previous Bb vaccination history indicate that a lifestyle that provides potential exposure to other dogs during visits to dog parks or wildlife reservoirs of Bb infection, such 862
as naturally infected rabbits (19) does not guarantee Bb exposure and resultant seropositivity. Relatedly, such dogs that are either naive to Bb or lacking in recent exposure may respond poorly to a single dose of a bacterin, as was the case in these dogs. In contrast, dogs with moderate to high concentrations of Bb-reactive antibody by virtue of previous vaccination or exposure may not produce significantly more antibody after vaccination. This is consistent with current concepts concerning the control of antibody responses (20). In this category were 3 dogs from 2 suburban households that had high concentrations of Bb-specific antibodies 6 to 9 mo after IN vaccination. Based on the predicted kinetics of antibody rise and decay (20) and previous studies of antibody kinetics in response to a single dose of IN Bb vaccine under conditions in which antigen exposure was controlled (12), it is likely that these dogs were somehow CVJ / VOL 55 / SEPTEMBER 2014
CVJ / VOL 55 / SEPTEMBER 2014
contrast, much less is known about the antibody specificity spectrum that develops in dogs following immunogenic exposure to Bb antigens (30). Our results provide preliminary evidence that a similar consetellation of antigen specific responses occurs in dogs after exposure to Bb antigens. Notably, as in Bp-vaccinated or Bp-infected humans (8,29), pertactin, which is a major bacterial adhesion molecule and varies in size depending on the strain of Bp or Bb (8,30), appears to be immunodominant in vaccinated or infected dogs. As in the Bp model (8,29), the current acellular vaccines in the respective target species also induce pertactin-specific responses, and to a lesser extent FHA similar to those seen with the previously used whole cell bacterins. In humans, antibody responses to both proteins are associated with disease sparing (8,29). In addition, antibody responses against p68 have disease sparing effects in Bb-infected pigs (31). Given the high degree of genetic, antigenic, and biological similarity between Bp and Bb (8,9), it is highly likely that IgG responses to these proteins are associated with clinical immunity in infected dogs as well. The specific identification and role of responses to the other proteins in the acellular Bb vaccine await further investigation. In human medicine an evolution away from whole cell Bp vaccines towards acellular or subunit products was justified on the basis of adverse “vaccine reactions” (up to 50% incidence) to whole cell Bp vaccines (8). A similar trend has occurred in Bb products used for dogs. In human medicine a very successful Bp whole cell bacterin has been replaced with various subunit vaccines that contain arbitrary amounts of 3 or 4 purified monomorphic Bp proteins (8). There are population “risks” for unquestioned pursuit of “defined” immunogens at the expense of effective, but biologically more complex products such as whole cell bacterins. The recent re-emergence of whooping cough and associated disease, and even death in “at risk” human populations highlights the potential fallacy of this reductionist approach to immunization (32,33). It is now well-documented that the current acellular Bp vaccines confer a shorter duration of immunity than did the previously used whole cell bacterins (33). More ominous, is documentation of the emergence of pertactin negative virulent strains in recent outbreaks of whooping cough that have markedly decreased “susceptibility” to the immune responses induced by the current subunit Bp vaccines (34). The latter phenomenon is thought to derive, at least in part, from the altered immunogenicity and immunodominance of constituents in the current vaccines, inducing antibody responses that apparently drive bacterial evolution (34). In spite of the unspoken belief within the biologics and regulatory communities that a simple and defined immunogen is the “best” vaccine candidate, the acellular Bb vaccine product evaluated in this study is less refined than the Bp products discussed above and still contains a broad constellation of native Bordetella antigens, responses to which have been associated with disease-sparing (8,31). Our data indicate that this product induces a broader range of Bb antigen-specific responses unlike the potentially deficient responses induced in humans with comparable acellular Bp vaccines. As well, the broad range antigen specific-responses that we report here occurred in response to an acellular vaccine with very low total protein concentration and no exogenous 863
A R T I C LE
naturally exposed to Bb (or Bp) antigens in the interval between IN vaccination and enrollment in the study. Overall, the results from the client-owned dogs demonstrate the marked variation in exposure and extant response to Bordetella spp. antigens that can occur in dogs in a representative veterinary practice. Moreover, this variation also highlighted the difficulty, in the absence of a vaccination history, of predicting a given dog’s serological/ exposure status at the time of presentation for vaccination and how it is likely to respond to a “booster shot.” Several studies have reported an association between both mucosal and systemic Bb antibody responses and subsequent disease sparing in Bb infected dogs and other species (4,5,7,10, 11,21). In dogs that received either intranasal modified live Bb or a parenteral whole cell bacterin, alone or in combination, the concentration of Bb-specific mucosal IgA and serum IgG was negatively correlated with bacterial load in both the nose and oropharynx of experimentally infected dogs (7). Moreover, this disease-sparing activity of a strong IgG response to parenteral vaccination is similar to those reported for injectable vaccineinduced serological responses in humans at risk for B. pertussis infections (8,22), including the possibility that individuals that have been primed by natural (subclinical) infection respond to parenteral immunization with not just an IgG response, but also with an IgA response (23). Results obtained in the bacterial killing assay, which is an in vitro model of IgG and complement mediated “immune elimination” (24), complement those findings, although there was not complete correspondence between increases in antibody after vaccination and killing of bacteria in vitro. It is likely that antibodies that bind to the 68-kD protein (pertactin), are responsible for bactericidal activity (25,26). The variability that we observed in antibody/C-mediated killing likely reflects the reported complexity of Bp-complement interactions that are described in humans (26). Both Bp and Bb possess a virulence factor, BrkA, a 103 kD transmembrane protein. This protein inhibits complement activation after binding/inactivating activated complement component C1, critical to completion of complement activation via the classical complement pathway (27). Curiously, some individuals mount an immune response to this protein that counteracts this effect, others do not (27,28). High concentrations of Bordetella-reactive IgA or subtypes of IgG, which do not activate complement can also “compete” for binding sites on bacterial surfaces, thereby blocking the IgG-C killing (28). These IgG subclass or IgA blocking effects are dependent upon the concentrations of the particular antibodies, and may vary with individual immune responses to vaccination versus infection (28). Whether these mechanisms play a role in modulating Bb bactericidal activity in dogs requires further investigation. In humans, the antigen specificity of the Bp-reactive antibody responses to infection and vaccination is well-documented (8,22,29). These responses depend somewhat on the formulation of the vaccine used in the case of vaccinates. A typical human response to Bp includes the development of antibodies that bind to the pertussis toxin (not expressed in Bb), the 220-kD fimbrial hemagglutinin (FHA), the 68 to 69-kD pertactin (PRN), various fimbrial antigens, the adenylate cyclase toxin, Bp lipopolysaccharide, unidentified molecules of 75 and 84 kD (8,29). In
adjuvant. These unique features should minimize the potential for adverse reactions to this vaccine when administered alone or in combination with other vaccines (35,36).
A R T I C LE
Acknowledgments The authors thank Dr. Yolande Miles and colleagues at the Due South Animal Hospital, Calgary, Alberta, for the provision of sera from client-owned dogs. This work was supported by the principal author’s discretionary funds to study viral immunology. CVJ
References 1. Ferry NS. A preliminary report of bacterial findings in canine distemper. Am Vet Rev 1910;37:499–504. 2. M’Gowan JP. Some observations on a laboratory epidemic, principally among dogs and cats in which the animals affected presented the symptoms of the disease called “distemper.” J Pathol Bacteriol 1911; 15:372–426. 3. Ellis JA, Anseeuw E, Gow S, et al. Seroepidemiology of respiratory (group 2) canine coronavirus, canine parainfluenza virus and Bordetella bronchiseptica infections in urban dogs in a humane shelter and in rural dogs in small communities. Can Vet J 2011;52:861–868. 4. McCandlish IA, Thompson H. Vaccination against canine bordetellosis using an aluminum hydroxide adjuvant vaccine. Res Vet Sci 1978; 25:51–57. 5. Shade FJ, Goodnow RA. Intranasal immunization of dogs against Bordetella bronchiseptica-induced tracheobronchitis (kennel cough) with modified-live Bordetella bronchiseptica vaccine. Am J Vet Res 1979;40:1241–1243. 6. Kontor EJ, Wegryzn MA, Goodnow RA. Canine infectious trachobronchitis: Effects of an intranasal live canine parainfluenza-Bordetella bronchiseptica vaccine on viral shedding and clinical tracheobronchitis (kennel cough). Am J Vet Res 1981;42:1694–1698. 7. Ellis JA, Haines DM, West KH, et al. Effect of vaccination on experimental infection with Bordetella bronchiseptica dogs. J Am Vet Med Assoc 2001;218:367–375. 8. Mattoo S, Cherry JD. Molecular pathogenesis, epidemiology, clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Micro Rev 2005;18:326–382. 9. Bjornstad ON, Harvill ET. Evolution and emergence of Bordetella in humans. Trends in Microbiol 2005;13:355–359. 10. Shade FJ, Rapp VJ. Studies of a bacterin incorporating an extracted Bordetella bronchiseptica antigen for controlling canine bordetellosis. Vet Med/Sm Anim Clin 1982:1635–1639. 11. Stephenson EH, Trahan CJ, Ezzell JW, et al. Efficacy of a commercial bacterin in protecting Strain 13 guineapigs against Bordetella bronchiseptica pneumonia. Lab An 1989;23:261–269. 12. Ellis JA, Krakowka GS, Dayton AD, Konoby C. Comparative efficacy of an injectable vaccine and an intranasal vaccine in stimulating Bordetella bronchiseptica-reactive antibody responses in seropositive dogs. J Am Vet Med Assoc 2002;220:43–48. 13. Davis R, Jayappa H, Abdelmagid OY, Armstrong R, Sweeney D, Lehr C. Comparison of the mucosal immune response in dogs vaccinated with either an intranasal avirulent live culture or a subcutaneous antigen extract vaccine of Bordetella bronchiseptica. Vet Therapeutics 2007;8:32–40. 14. Callister SM, Jobe DA, Agger WA, et al. Ability of the borreliacidal antibody test to confirm Lyme disease in clinical practice. Clin Diagn Lab Immunol 2002;9:908–912. 15. Stevens MG, Kehrli ME, Canning PC. A colorimetric assay for quantitating bovine neutrophil bactericidal activity. Vet Immunol Immunpathol 1991;28:45–56.
864
16. Ellis JA, Hawk DA, Mills KW, Pratt DL. Antigen specificity and activity of ovine antibodies induced by immunization with Corynebacterium pseudotuberculosis culture filtrate. Vet Immunol Immunopathol 1991;28:303–316. 17. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–254. 18. Switzer RC, Merril CR, Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem 1979;98:231–237. 19. Pathak AK, Creppage KE, Werner JR, Cattadori IM. Immune regulation of a chronic bacterial infection and consequences for pathogen transmission. BMC Microbiol 2010;10:226. 20. Tizzard IR. B cells and their response to antigens. In: Veterinary Immunology: An Introduction. 8th ed. St. Louis, Missouri: Saunders Elsevier, 2009:152–169. 21. Goodnow RA, Shade FJ, Switzer WP. Efficacy of Bordetella bronchiseptica bacterin in controlling enzootic atrophic rhinitis in swine. Am J Vet Res 1979;40:58–60. 22. Cherry JD, Gorbein J, Heininger U, Stehr K. A search for serological correlates of immunity to Bordetella pertussis cough illness. Vaccine 1998;16:1901–1906. 23. Le T, Cherry JD, Chang SJ, et al. Immune responses and antibody decay following immunization of adolescents and adults with an acellular pertussis vaccine: APERT study. J Infect Dis 2004;190:535–544. 24. Tizzard IR. Immunity at body surfaces. In: Veterinary Immunology: An Introduction. 8th ed. St. Louis, Missouri: Saunders Elsevier, 2009:245–248. 25. Gotto JW, Eckhardt T, Reilly PA. Biochemical and immunological properties of two forms of pertactin, the 69 000-molecularweight outer membrane protein of Bordetella pertussis. Infect Immun 1993;61:2211–2215. 26. Weiss AA, Patton AK, Millen SH, Chang SJ, Ward JI, Bernstein DI. Acellular pertussis vaccines and complement killing of Bordetella pertussis. Infect Immun 2004;72:7346–7351. 27. Barnes MG, Weiss AA. BrkA protein of Bordetella pertussis inhibits the classical pathway of complement after C1 deposition. Infect Immun 2001;69:3067–3072. 28. Griffiss JM, Goroff DK. IgA blocks IgM and IgG-initiated immune lysis by separate moleucular mechanisms. J Immunol 1983;130:2882–2885. 29. Redd SC, Rumschlag HS, Biellik RJ, Sanden GN, Reimer CB, Cohen ML. Immunoblot analysis of humoral immune responses following infection with Bordetella pertussis or immunization with diphtheriatetanus-pertussis vaccine. J Clin Microbiol 1988;26:1373–1377. 30. Keil DJ, Fenwick B. Strain- and growth condition-dependent variability in outer membrane protein expression by Bordetella bronchiseptica isolates from dogs. Am J Vet Res 1999;60:1016–1021. 31. Kobisch M, Novotny P. Identification of a 68-kilodalton outer membrane protein as the major protective antigen of Bordetella bronchiseptica by using specific-pathogen-free piglets. Infect Immun 1990;58:352–357. 32. Cherry JD. Epidemic pertussis in 2012 — The resurgence of a vaccinepreventable disease. New Eng J Med 2012;367:785–787. 33. Klein NP, Bartlett J, Rowhani-Rahbar A, Fireman B, Baxter R. Waning protection after fifth dose of acellular pertussis vaccine in children. New Eng J Med 2012;367:1013–1019. 34. Queenan AM, Cassiday PK, Evangelista A. Pertactin-negative variants of Bordetella pertussis in the United States. New Eng J Med 2012;368:583–584. 35. Moore GE, Guptil LF, Ward MP, et al. Adverse events diagnosed within three days of vaccine administration in dogs. J Am Vet Med Assoc 2007; 227:1102–1108. 36. Tizzard IR. The use of vaccines. In: Veterinary Immunology: An Introduction. 8th ed. St. Louis, Missouri: Saunders Elsevier, 2009: 270–276.
CVJ / VOL 55 / SEPTEMBER 2014
